The receptor-type tyrosine-protein phosphatase C, also known as PTPRC, is an enzyme that, in humans, is encoded by the PTPRC gene. PTPRC is also known as CD45 antigen (SEQ ID NO:29). The family of protein tyrosine phosphatases are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPRC (CD45 antigen) contains an extracellular domain including two fibronectin type-III domains, a single transmembrane segment and a cytoplasmic domain with two tyrosine-protein phosphatase domains. The first phosphatase domain has enzymatic activity, while the second one seems to affect the substrate specificity of the first one. PTPRC (CD45 antigen) is heavily N- and O-glycosylated, having 19 published N-glycosylation sites (UniProt entry P08575, see world wide web at uniprot.org). PTPRC (CD45 antigen) is expressed in leukocytes. CD45R0 (sometimes also written as CD45RO) is a splice variant or isoform of CD45 antigen, which has a shortened extracellular domain containing mainly the fibronectin type-III domains due to removal of exons 4-6 (which encode RA, RB and RC). Other splice variants or isoforms comprise CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, and CD45R (ABC). CD45R0 is expressed in subsets of T-cells such as activated and memory T-cells, in subsets of B-cells, monocytes, macrophages, and in subsets of natural killer cells in patients with an NK lymphocytosis. CD45RA is expressed in B-cells, naïve T-cells, and monocytes. CD45RB is expressed in subsets of T-cells, B-cells, monocytes, macrophages, and granulocytes. Due to the many different isoforms, the high degree of glycosylation and the short extracellular domain of CD45R0 antigen, it is complicated to generate functionally active and specific antibodies and fragments thereof against CD45R0. The murine antibody clone UCHL1 (Smith et al. (1986) Immunology 58, 63-70) is the most commonly used functional antibody against the human cell surface antigen CD45R0. The exact epitope on CD45R0 to which UCHL1 binds remains unclear, but it is destroyed by treatment with either neuraminidase or O-glycosidase (Pulido et al. (1994) J Exp Med 179, 1035-1040).
Antibodies against CD45R0 can be used for positive selection/enrichment or depletion of CD45R0-positive cells. Humanized antibodies or fragments thereof are useful for clinical and cell therapeutic purposes as they are less immunogenic when administered to human patients compared to non-humanized antibodies or fragments thereof such as murine antibodies or fragments thereof. Techniques for humanization of non-human antibodies are known-in-the-art and comprise complementary determining regions (CDR) grafting into a human or humanized framework, wherein the grafted CDRs are typically derived from a rodent antibody. CDRs are part of the variable regions in antibodies or fragments thereof, where these molecules bind to their specific antigen. They are embedded into framework regions which are much less variable than the CDRs.
There are several approaches to select an appropriate framework. In one approach, a well-known and field-tested framework can be used which already has been shown to generate functional antibodies and fragments thereof which can be expressed at high level. In another approach, a human framework sequence is selected from a group of germline sequences which shows a high degree of identity to the original framework amino acid sequence (“germline humanization”; Ahmadzadeh et al. (2014), Protein Expression and Purification 102, 45-51). In some cases, the CDR sequences are additionally included to the framework sequence for the selection of a human framework with a high degree of identity. Depending on the amino acid sequence of the antibody to be humanized, the highest degree of identity of a germline framework sequence to the original framework sequence may be as low as 50% for the heavy chain variable domain and 40% for the light chain variable domain. Depending on the exact sequence used for the determination of the germline framework sequence with highest identity, for example including or excluding the CDR regions, the resulting sequences may differ from each other, meaning that more than one framework sequence may exist that per definition has the highest degree of identity to the original framework amino acid sequence.
Compared with framework regions derived from mature IgGs, the germline genes have less intraclonal somatic hypermutations. Therefore, it is expected that humanized antibodies derived from germline frameworks show lower immunogenicity than humanized antibodies with mature IgG frameworks.
In many cases, the affinity of antibodies engineered by CDR grafting is significantly reduced. Some rodent residues in framework regions, referred to as Vernier zone residues, have been demonstrated to affect the conformation of CDR loops and affinity of antibodies. These residues are located in the beta-sheet framework regions closely underlying the CDRs. Therefore, after the selection of desired human frameworks these residues are preferably maintained in humanized antibodies (Safdari et al. (2013) Biotechnology and Genetic Engineering Reviews 29, 175-186). The change of a human Vernier zone residue back to the original rodent residue at the respective position within the antibody variable regions is referred to as a “backmutation”. Techniques for backmutations and the selection of amino acid residues to be backmutated are known-in-the-art and comprise structure-based approaches as well as random mutagenesis.
EP1421191 discloses humanized antibodies against CD45 antigen isoforms. The described antibody binds to CD45R0 and to CD45RB, whereas antibody clone UCHL1 specifically binds CD45R0. The specific binding of UCHL1 was published by Terry et al. (1988) Immunol. 64, 331-336, showing that antibodies such as UCHL1 selectively bind to the 180 kDa isoform CD45R0 (without any of the variable exons A, B or C) which appears to be restricted to a subset of activated T cells, memory cells and cortical thymocytes and is not detected on B cells.
It does not seem to exist a humanized antibody or fragment thereof against CD45 which only binds to the CD45R0 isoform of the antigen. Therefore, there is a need in the art for a humanized antibody or fragment thereof specific for the CD45R0 isoform of the CD45 antigen which can be used for clinical and cell therapeutic purposes, for example as part of a clinical cell separation reagent.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.